Glyphosate resistant class i 5-endolpyruvylshikimate-3-phosphate synthase (epsps)

ABSTRACT

The compositions and methods disclosed herein provide novel DNA molecules that encode glyphosate resistant EPSPS proteins and plants containing these new proteins. The plants that express the new EPSPS proteins are themselves tolerant to the herbicidal effects of glyphosate.

This application claims benefit of U.S. Provisional Application No.60/448,438, filed Feb. 18, 2003, the entire contents of which areincorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to plant molecular biology and plant geneticengineering for herbicide resistance and, more particularly, to class I5-enolpyruvylshikimate-3-phosphate synthases modified for glyphosateresistance. Plant genetic engineering methods are used to modify class I5-enolpyruvylshikimate-3-phosphate synthase DNA and the encodedproteins, and to transfer these molecules into plants of agronomicimportance. More specifically, the invention comprises DNA and proteincompositions of glyphosate resistant 5-enolpyruvylshikimate-3-phosphatesynthases, and to the plants containing these compositions.

BACKGROUND OF THE INVENTION

N-phosphonomethylglycine, also known as glyphosate, is a well-knownherbicide that has activity on a broad spectrum of plant species.Glyphosate is the active ingredient of Roundup® (Monsanto Co., St Louis,Mo.), a herbicide having a long history of safe use and a desirablyshort half-life in the environment. When applied to a plant surface,glyphosate moves systemically through the plant. Glyphosate isphytotoxic due to its inhibition of the shikimic acid pathway, whichprovides a precursor for the synthesis of aromatic amino acids.Glyphosate inhibits the class I 5-enolpyruvyishikimate-3-phosphatesynthase (EPSPS) found in plants and some bacteria. Glyphosate tolerancein plants can be achieved by the expression of a modified class I EPSPSthat has lower affinity for glyphosate, yet still retains its catalyticactivity in the presence of glyphosate (U.S. Pat. Nos. 4,535,060, and6,040,497). “Tolerant” or “tolerance” refers to a reduced effect of anagent on the growth and development, and yield of a plant, inparticular, tolerance to the phytotoxic effects of a herbicide,especially glyphosate.

Enzymes, such as, class II EPSPSs have been isolated from bacteria thatare naturally resistant to glyphosate and when the enzyme is expressedas a transgene in plants provides glyphosate tolerance to the plants(U.S. Pat. Nos. 5,633,435 and 5,094,945). Enzymes that degradeglyphosate in plant tissues (U.S. Pat. No. 5,463,175) are also capableof conferring plant tolerance to glyphosate. DNA constructs that containthe necessary genetic elements to express the glyphosate resistantenzymes or degradative enzymes create chimeric transgenes useful inplants. Such transgenes are used for the production of transgenic cropsthat are tolerant to glyphosate, thereby allowing glyphosate to be usedfor effective weed control with minimal concern of crop damage. Forexample, glyphosate tolerance has been genetically engineered into corn(U.S. Pat. No. 5,554,798), wheat (Zhou et al. Plant Cell Rep.15:159-163, 1995), soybean (WO 9200377) and canola (WO 9204449). Thetransgenes for glyphosate tolerance and transgenes for tolerance toother herbicides, for example the bar gene (Sh.Bar) may be included inDNA constructs for use as a selectable marker for plant transformation(present invention pMON81519; and Toki et al. Plant Physiol.,100:1503-1507, 1992; Thompson et al. EMBO J. 6:2519-2523, 1987;phosphinothricin acetyltransferase DeBlock et al. EMBO J., 6:2513-2522,1987, glufosinate herbicide) are also useful as selectable markers orscorable markers and can provide a useful phenotype for selection oftransgenic plants when the marker gene is linked with otheragronomically useful traits.

Development of herbicide-tolerant crops has been a major breakthrough inagriculture biotechnology as it has provided farmers with new weedcontrol methods. One enzyme that has been successfully engineered forresistance to its inhibitor herbicide is class I EPSPS. Variants ofclass I EPSPS have been isolated (Pro-Ser, U.S. Pat. No. 4,769,061;Gly-Ala, U.S. Pat. No. 4,971,908; Gly-Ala, Gly-Asp, U.S. Pat. No.5,310,667; Gly-Ala, Ala-Thr, U.S. Pat. No. 5,8866,775) that areresistant to glyphosate. However, many EPSPS variants either do notdemonstrate a sufficiently high K_(i) for glyphosate or have a K_(m) forphosphoenol pyruvate (PEP) too high to be effective as a glyphosateresistance enzyme for use in plants (Padgette et. al, In“Herbicide-resistant Crops”, Chapter 4 pp 53-83. ed. Stephen Duke, LewisPub, CRC Press Boca Raton, Fla. 1996). However, one class I EPSPSvariant, T102I/P106S (TIPS) that is operably linked to a heterologouspromoter has been shown to provide glyphosate tolerance to transgenicmaize plants (U.S. Pat. No. 6,040,497). A glyphosate tolerant EPSPS hasalso been isolated from the weed Eleusine indica [WO 01/66704].

There is a need in the field of plant molecular biology for a diversityof genes that can provide a positive selectable marker phenotype. Inparticular, glyphosate tolerance is used extensively as a positiveselectable marker in plants and is a valuable phenotype for use in cropproduction. The stacking and combining of existing transgene traits withnewly developed traits is enhanced when distinct positive selectablemarker genes are used. The marker genes provide either a distinctphenotype, such as, antibiotic or herbicide tolerance, or a moleculardistinction discernable by methods used for DNA detection. Thetransgenic plants can be screened for the stacked traits by analysis formultiple antibiotic or herbicide tolerance or for the presence of novelDNA molecules by DNA detection methods. The present invention providesDNA and protein compositions of glyphosate resistant variant class IEPSP synthases. The present invention also provides DNA constructsuseful in plants and transgenic plants that exhibit glyphosatetolerance.

SUMMARY OF THE INVENTION

In one aspect of the invention there is provided an isolated modifiedEPSPS DNA molecule encoding a glyphosate tolerant EPSPS protein havingan isoleucine or leucine at position 102, and an amino acid at position106 selected from the group consisting of threonine, glycine, cysteine,alanine, and isoleucine. In another aspect of the invention is a DNAconstruct that comprises a promoter that functions in plant cellsoperably linked to a modified EPSPS DNA molecule encoding a glyphosatetolerant EPSPS protein having an isoleucine or leucine at position 102,and an amino acid at position 106 selected from the group consisting ofthreonine, glycine, cysteine, alanine, and isoleucine. In yet anotheraspect of the invention there is provided a transgenic plant thancontains the DNA construct, wherein the transgenic plant is tolerant toglyphosate herbicide.

In another aspect of the invention is a method of preparing a fertiletransgenic plant comprising providing a plant expression cassette havinga modified EPSPS gene encoding an EPSPS protein having isoleucine orleucine at position 102, and an amino acid at position 106 selected fromthe group consisting of threonine, glycine, cysteine, alanine, andisoleucine; and contacting recipient plant cells with the plantexpression cassette under conditions permitting the uptake of the plantexpression cassette by the recipient cells; and selecting the recipientplant cells that contain the plant expression cassette; and regeneratingplants from the selected recipient plant cells; and identifying afertile transgenic plant that is tolerant to glyphosate.

In another aspect of the invention is a fertile glyphosate toleranttransgenic plant that contains a plant expression cassette having amodified plant EPSPS gene encoding an EPSPS protein having isoleucine orleucine at position 102, and an amino acid at position 106 selected fromthe group consisting of threonine, glycine, cysteine, alanine, andisoleucine that is crossed to another plant to provide progeny that aretolerant to glyphosate.

In another aspect of the invention, there is provided a method forcontrolling weeds in a field of crop plants, wherein the field of cropplants is treated with an effective amount of a glyphosate containingherbicide and the crop plants contain a plant expression cassette havinga modified EPSPS gene encoding an EPSPS protein having isoleucine orleucine at position 102, and an amino acid at position 106 selected fromthe group consisting of threonine, glycine, cysteine, alanine, andisoleucine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Polynucleotide and polypeptide sequence of maize EPSPS.

FIG. 2. Polypeptide alignment of class I plant and bacterial EPSPsynthases.

FIG. 3. DNA construct map of pMON70461 (wild-type EPSPS).

FIG. 4. DNA primer sequences

FIG. 5. DNA construct map of pMON58452 (ZmTIPT variant)

FIG. 6. DNA construct map of pMON30167 (CP4 EPSPS)

FIG. 7. DNA construct map of pMON70472 (ZmTIPT variant)

FIG. 8. DNA construct map of pMON70475 (ZmTIPA variant)

FIG. 9. DNA construct map of pMON70467 (ZmPT variant)

FIG. 10. DNA construct map of pMON81519 (ZmTIPT variant)

FIG. 11. DNA construct map of pMON81548 (LsTIPA variant)

FIG. 12. DNA construct map of pMON58491 (AtTIPA variant)

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the construction of aglyphosate resistant EPSPS and utilizing DNA molecules that encode theEPSPS in a DNA construct to provide herbicide tolerance to transgenicplants expressing the glyphosate resistant EPSPS in its tissues. Thefollowing descriptions are provided to better define the presentinvention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag: New York, (1991); and Lewin,Genes V, Oxford University Press: New York, (1994). The nomenclature forDNA bases as set forth at 37 CFR §1.822 is used. “Nucleic acid” refersto deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The standardone- and three-letter nomenclature for amino acid residues is used.

Methods of the present invention include designing EPSPS proteins thatconfer a glyphosate tolerant trait to the plant into which they areintroduced. Polynucleotide molecules encoding proteins involved inherbicide tolerance are known in the art, and include, but are notlimited to a polynucleotide molecule encoding5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, described in U.S.Pat. Nos. 5,627,061, 5,633,435, and 6,040,497; Padgette et al. HerbicideResistant Crops, Lewis Publishers, 53-85, 1996; and Penaloza-Vazquez, etal. Plant Cell Reports 14:482-487, 1995; and aroA (U.S. Pat. No.5,094,945) for glyphosate tolerance.

“Glyphosate” refers to N-phosphonomethylglycine and its' salts.Glyphosate is the active ingredient of Roundup® herbicide (MonsantoCo.). Plant treatments with “glyphosate” refer to treatments with theRoundup® or Roundup Ultra® herbicide formulation, unless otherwisestated. Glyphosate as N-phosphonomethylglycine and its' salts (notformulated Roundup® herbicide) are components of synthetic culture mediaused for the selection of bacteria and plant tolerance to glyphosate orused to determine enzyme resistance in in vitro biochemical assays.Examples of commercial formulations of glyphosate include, withoutrestriction, those sold by Monsanto Company as ROUNDUP®, ROUNDUP® ULTRA,ROUNDUP® ULTRAMAX, ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP® BIACTIVE,ROUNDUP® BIOFORCE, RODEO®, POLARIS®, SPARK® and ACCORD® herbicides, allof which contain glyphosate as its isopropylammonium salt; those sold byMonsanto Company as ROUNDUP® DRY and RIVAL® herbicides, which containglyphosate as its ammonium salt; that sold by Monsanto Company asROUNDUP® GEOFORCE, which contains glyphosate as its sodium salt; andthat sold by Zeneca Limited as TOUCHDOWN® herbicide, which containsglyphosate as its trimethylsulfonium salt.

Through plant genetic engineering methods, it is possible to produceglyphosate tolerant plants by inserting into the plant genome a DNAmolecule that causes the production of higher levels of wild-type EPSPS(U.S. Pat. No. 4,940,835; Shah et al., Science 233:478-481, 1986).Glyphosate tolerance can also be achieved by the expression of EPSPSvariants that have lower affinity for glyphosate and therefore retaintheir catalytic activity in the presence of glyphosate, for example,aroA P-S (U.S. Pat. No. 5,094,945), CP4 EPSPS (U.S. Pat. No. 5,633,435),maize TIPS (U.S. Pat. No. 6,040,497), 101/192 and 101/144 variants (U.S.Pat. No. 5,866,775 and U.S. Pat. No. 6,225,112, Howe et al., Mol.Breeding. 10:153-164, 2002). For example, glyphosate tolerance has beengenetically engineered into corn (U.S. Pat. Nos. 5,554,798, 6,040,497),wheat (Zhou et al. Plant Cell Rep. 15:159-163, 1995), soybean (WO9200377), cotton (WO 0234946), and canola (WO 9204449).

Variants of the wild-type EPSPS enzyme have been isolated that areglyphosate-resistant as a result of alterations in the EPSPS amino acidcoding sequence (Kishore et al., Annu. Rev. Biochem. 57:627-663, 1988;Schulz et al., Arch. Microbiol. 137:121-123, 1984; Sost et al., FEBSLett. 173:238-241, 1984; Kishore et al., In “Biotechnology for CropProtection” ACS Symposium Series No. 379. eds. Hedlin et al., 37-48,1988). These variants typically have a higher K_(i) for glyphosate thanthe wild-type EPSPS enzyme that confers the glyphosate-tolerantphenotype, but these variants are also characterized by a high K_(m) forPEP that makes the enzyme kinetically less efficient. For example, theapparent K_(m) for PEP and the apparent K_(i) for glyphosate for thenative EPSPS from E. coli are 10 μM and 0.5 μM while for aglyphosate-resistant isolate having a single amino acid substitution ofan alanine for the glycine at position 96 these values are 220 μM and4.0 mM, respectively. U.S. Pat. No. 6,040,497 reports that the EPSPSvariant, known as the TIPS mutation (a substitution of isoleucine forthreonine at amino acid position 102 and a substitution of serine forproline at amino acid position 106) comprises two mutations that whenintroduced into the polypeptide sequence of Zea mays EPSPS confersglyphosate resistance to the enzyme. Transgenic plants containing thismutant enzyme are tolerant to glyphosate. Identical mutations may bemade in the genes encoding glyphosate sensitive EPSPS enzymes from othersources to create glyphosate resistant enzymes. In vitro site-directedmutagenesis of DNA molecules have clearly demonstrated utility forintroducing specific changes in a DNA sequence of a genome and othermethods under development may also provide in situ site-directedmutagenesis methods (US Patent Pub. 20020151072). These methods may beused to generate the DNA coding sequences that encode for the glyphosateresistant EPSPS variants of the present invention in the context of theendogenous host cell EPSPS gene.

The present invention provides amino acid substitutions in a class IEPSPS that demonstrates enhanced glyphosate resistance over anypreviously described modified class I EPSPSs. The present inventionrelates specifically to certain double variants of class I EPSPSs thatare glyphosate resistant, but still retain a functional level of PEPsubstrate binding activity. During the development of the novel doublevariants of class I EPSPSs for glyphosate resistance, it was necessaryto construct a number of single variants useful as controls for theassay and for demonstration that the double variant is necessary toobtain both a glyphosate resistant enzyme and an enzyme that stillretains a sufficient level of substrate binding activity to serve as afunctional replacement for a native class I EPSPS.

The EPSPS enzyme functions in plant chloroplast, therefore, chloroplasttransit peptides (CTP) are engineered in a DNA molecule to encode afusion of the CTP to the N terminus of an EPSPS creating a chimericmolecule. A chimeric polynucleic acid coding sequence is comprised oftwo or more open reading frames joined in-frame that encode a chimericprotein, for example, a chloroplast transit peptide and an EPSPS enzyme.A chimeric gene refers to the multiple genetic elements derived fromheterologous sources operably linked to comprise a gene. The CTP directsthe glyphosate resistant enzyme into the plant chloroplast. In thenative plant EPSPS gene, chloroplast transit peptide regions arecontained in the native coding sequence (for example, CTP2, Klee et al.,Mol. Gen. Genet. 210:47-442, 1987). The CTP is cleaved from the EPSPSenzyme at the chloroplast membrane to create a “mature EPSPS or EPSPSenzyme” that refers to the polypeptide sequence of the processed proteinproduct remaining after the chloroplast transit peptide has beenremoved.

The native CTP may be substituted with a heterologous CTP duringconstruction of a transgene plant expression cassette. Manychloroplast-localized proteins, including EPSPS, are expressed fromnuclear genes as precursors and are targeted to the chloroplast by achloroplast transit peptide (CTP) that is removed during the importsteps. Examples of other such chloroplast proteins include the smallsubunit (SSU) of ribulose-1,5-bisphosphate carboxylase (rubisco),ferredoxin, ferredoxin oxidoreductase, the light-harvesting complexprotein I and protein H, and thioredoxin F. It has been demonstrated invivo and in vitro that non-chloroplast proteins may be targeted to thechloroplast by use of protein fusions with a CTP and that a CTP sequenceis sufficient to target a protein to the chloroplast. Incorporation of asuitable chloroplast transit peptide, such as, the Arabidopsis thalianaEPSPS CTP (Klee et al., Mol. Gen. Genet. 210:437-442 (1987), and thePetunia hybrida EPSPS CTP (della-Cioppa et al., Proc. Natl. Acad. Sci.USA 83:6873-6877 (1986) has been shown to target heterologous EPSPSprotein to chloroplasts in transgenic plants. The production ofglyphosate tolerant plants by expression of a fusion protein comprisingan amino-terminal CTP with a glyphosate resistant EPSPS enzyme is wellknown by those skilled in the art, (U.S. Pat. No. 5,627,061, U.S. Pat.No. 5,633,435, U.S. Pat. No. 5,312,910, EP 0218571, EP 189707, EP508909, and EP 924299). Those skilled in the art will recognize thatvarious chimeric constructs can be made that utilize the functionalityof a particular CTP to import glyphosate resistant EPSPS enzymes intothe plant cell chloroplast.

Modification and changes may be made in the structure of the DNApolynucleotides of the invention and still obtain a DNA molecule thattranscribes a mRNA that encodes the modified functional EPSPS protein ofthe present invention. The amino acid substitutions disclosed hereinprovide an improved characteristic to the protein, for example, enhancedglyphosate resistant EPSP synthase. Amino-acid substitutions oramino-acid variants, are preferably substitutions of a single amino-acidresidue for another amino-acid residue at one or more positions withinthe protein. Substitutions, deletions, insertions or any combinationthereof can be combined to arrive at a final construct. The presentinvention involves the substitution of amino acids in a class I EPSPSprotein to provide a new feature of the protein, such as, glyphosateresistance.

It is known that the genetic code is degenerate. The amino acids andtheir RNA codon(s) are listed below in Table 1.

TABLE 1 Amino acids and the RNA codons that encode them. Amino Acid Fullname; 3 letter code; 1 letter code Codons Alanine; Ala; A GCA GCC GCGGCU Cysteine; Cys; C UGC UGU Aspartic acid; Asp; D GAC GAU Glutamicacid; Glu; E GAA GAG Phenylalanine; Phe; F UUC UUU Glycine; Gly; G GGAGGC GGG GGU Histidine; His; H CAC CAU Isoleucine; Ile; I AUA AUC AUULysine; Lys; K AAA AAG Leucine; Leu; L UUA UUG CUA CUC CUG CUUMethionine; Met; M AUG Asparagine; Asn; N AAC AAU Proline; Pro; P CCACCC CCG CCU Glutamine; Gln; Q CAA CAG Arginine; Arg; R AGA AGG CGA CGCCGG CGU Serine; Ser; S AGC AGU UCA UCC UCG UCU Threonine; Thr; T ACA ACCACG ACU Valine; Val; V GUA GUC GUG GUU Tryptophan; Trp; W UGG Tyrosine;Tyr; Y UAC UAU

The codons are described in terms of RNA bases, for example adenine,uracil, guanine and cytosine, it is the mRNA that is directly translatedinto polypeptides. It is understood that when designing a DNApolynucleotide for use in a construct, the DNA bases would besubstituted, for example, thymine instead of uracil. Codon refers to asequence of three nucleotides that specify a particular amino acid.Codon usage or “codon bias” refers to the frequency of use of codonsencoding amino acids in the coding sequences of organisms. A codon usagetable would be consulted when selecting substituting codons for anartificial DNA sequence. The sequence of codons provides a codingsequence that refers to the region of continuous sequential nucleic acidtriplets encoding a protein, polypeptide, or peptide sequence. The term“encoding DNA” refers to chromosomal DNA, plasmid DNA, cDNA, orartificial DNA polynucleotide that encodes any of the proteins discussedherein. “Plasmid” refers to a circular, extrachromosomal,self-replicating piece of DNA.

The term “endogenous” refers to materials originating from within anorganism or cell. “Exogenous” refers to materials originating fromoutside of an organism or cell. This typically applies to nucleic acidmolecules used in producing transformed or transgenic host cells andplants.

The term “genome” as it applies to bacteria encompasses both thechromosome and plasmids within a bacterial host cell. Encoding nucleicacids of the present invention introduced into bacterial host cells cantherefore be either chromosomally-integrated or plasmid-localized. Theterm “genome” as it applies to plant cells encompasses not onlychromosomal DNA found within the nucleus, but organelle DNA found withinsubcellular components of the cell. The term “gene” refers topolynucleic acids that comprise chromosomal DNA, plasmid DNA, cDNA, anartificial DNA polynucleotide, or other DNA that is transcribed into anRNA molecule, wherein the RNA may encode a peptide, polypeptide, orprotein, and the genetic elements flanking the coding sequence that areinvolved in the regulation of expression of the mRNA or polypeptide ofthe present invention. A “fragment” of a gene is a portion of afull-length polynucleic acid molecule that is of at least a minimumlength capable of transcription into a RNA, translation into a peptide,or useful as a probe or primer in a DNA detection method.

Polynucleic acids of the present invention introduced into plant cellscan therefore be either chromosomally-integrated or organelle-localized.The modified EPSPSs of the present invention are targeted to thechloroplast by a chloroplast transit peptide located at the N-terminusof the coding sequence. Alternatively, the gene encoding the modifiedEPSPSs may be integrated into the chloroplast genome, therebyeliminating the need for a chloroplast transit peptide.

“Heterologous DNA” sequence refers to a polynucleotide sequence thatoriginates from a foreign source or species or, if from the same source,is modified from its original form. “Homologous DNA” refers to DNA fromthe same source as that of the recipient cell.

“Hybridization” refers to the ability of a strand of nucleic acid tojoin with a complementary strand via base pairing. Hybridization occurswhen complementary sequences in the two nucleic acid strands bind to oneanother. The nucleic acid probes and primers of the present inventionhybridize under stringent conditions to a target DNA sequence. Anyconventional nucleic acid hybridization or amplification method can beused to identify the presence of DNA from a transgenic event in asample. A transgenic “event” is produced by transformation of a plantcell with heterologous DNA, i.e., a nucleic acid construct that includesa transgene of interest; regeneration of a population of plantsresulting from the insertion of the transgene into the genome of theplant cell, and selection of a particular plant characterized byinsertion into a particular genome location. The term “event” refers tothe original transformant plant and progeny of the transformant thatinclude the heterologous DNA. The term “event” also includes progenyproduced by a sexual outcross between the event and another plant thatwherein the progeny includes the heterologous DNA. Nucleic acidmolecules or fragments thereof are capable of specifically hybridizingto other nucleic acid molecules under certain circumstances. As usedherein, two nucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure. Anucleic acid molecule is said to be the “complement” of another nucleicacid molecule if they exhibit complete complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the molecules is complementary to a nucleotide ofthe other. Two molecules are said to be “minimally complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to be“complementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., Molecular Cloning—ALaboratory Manual, 2nd. ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989), herein referred to as Sambrook et al., (1989), andby Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach,IRL Press, Washington, D.C. (1985). Departures from completecomplementarity are therefore permissible, as long as such departures donot completely preclude the capacity of the molecules to form adouble-stranded structure. In order for a nucleic acid molecule to serveas a primer or probe it need only be sufficiently complementary insequence to be able to form a stable double-stranded structure under theparticular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. The term “stringent conditions” is functionallydefined with regard to the hybridization of a nucleic-acid probe to atarget nucleic acid (such as, to a particular nucleic-acid sequence ofinterest) by the specific hybridization procedure discussed in Sambrooket al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at9.47-9.52, 9.56-9.58; Kanehisa, (Nucl. Acids Res. 12:203-213, 1984); andWetmur and Davidson, (J. Mol. Biol. 31:349-370, 1988). Accordingly, thenucleotide sequences of the invention may be used for their ability toselectively form duplex molecules with complementary stretches of DNAfragments. Depending on the application envisioned, one will desire toemploy varying conditions of hybridization to achieve varying degrees ofselectivity of probe towards target sequence. For applications requiringhigh selectivity, one will typically desire to employ relativelystringent conditions to form the hybrids, for example, one will selectrelatively low salt and/or high temperature conditions, such as providedby about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. toabout 70° C. A stringent condition, for example, is to wash thehybridization filter at least twice with high-stringency wash buffer(0.2×SSC, 0.1% SDS, 65° C.). Appropriate stringency conditions thatpromote DNA hybridization, for example, 6.0× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C.,are known to those skilled in the art or can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged. Such selective conditions tolerate little, if any, mismatchbetween the probe and the template or target strand. Detection of DNAmolecules via hybridization is well known to those of skill in the art,and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 areexemplary of the methods of hybridization analyses.

“Identity” refers to the degree of similarity between two polynucleicacid or protein sequences. An alignment of the two sequences isperformed by a suitable computer program. A widely used and acceptedcomputer program for performing sequence alignments is CLUSTALW v1.6(Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994). The number ofmatching bases or amino acids is divided by the total number of bases oramino acids, and multiplied by 100 to obtain a percent identity. Forexample, if two 580 base pair sequences had 145 matched bases, theywould be 25 percent identical. If the two compared sequences are ofdifferent lengths, the number of matches is divided by the shorter ofthe two lengths. For example, if there are 100 matched amino acidsbetween 200 and 400 amino acid proteins, they are 50 percent identicalwith respect to the shorter sequence. If the shorter sequence is lessthan 150 bases or 50 amino acids in length, the number of matches aredivided by 150 (for nucleic acid bases) or 50 (for amino acids), andmultiplied by 100 to obtain a percent identity.

“Intron” refers to a genetic element that is a portion of a gene nottranslated into protein, even though it is transcribed into RNA, theintron sequence being “spliced out” from the mature messenger RNA.

An “isolated” nucleic acid molecule is substantially separated away fromother nucleic acid sequences with which the nucleic acid is normallyassociated, such as, from the chromosomal or extrachromosomal DNA of acell in which the nucleic acid naturally occurs. A nucleic acid moleculeis an isolated nucleic acid molecule when it comprises a transgene orpart of a transgene present in the genome of another organism. The termalso embraces nucleic acids that are biochemically purified so as tosubstantially remove contaminating nucleic acids and other cellularcomponents. The term “transgene” refers to any polynucleic acid moleculenormative to a cell or organism transformed into the cell or organism.“Transgene” also encompasses the component parts of a native plant genemodified by insertion of a normative polynucleic acid molecule bydirected recombination or site specific mutation.

“Isolated,” “Purified,” “Homogeneous” polypeptides. A polypeptide is“isolated” if it has been separated from the cellular components(nucleic acids, lipids, carbohydrates, and other polypeptides) thatnaturally accompany it or that is chemically synthesized or recombinant.A polypeptide molecule is an isolated polypeptide molecule when it isexpressed from a transgene in another organism. A monomeric polypeptideis isolated when at least 60% by weight of a sample is composed of thepolypeptide, preferably 90% or more, more preferably 95% or more, andmost preferably more than 99%. Protein purity or homogeneity isindicated, for example, by polyacrylamide gel electrophoresis of aprotein sample, followed by visualization of a single polypeptide bandupon staining the polyacrylamide gel; high pressure liquidchromatography; or other conventional methods. Proteins can be purifiedby any of the means known in the art, for example as described in Guideto Protein Purification, ed. Deutscher, Meth. Enzymol. 185, AcademicPress, San Diego, 1990; and Scopes, Protein Purification: Principles andPractice, Springer Verlag, New York, 1982.

The term “native” generally refers to a naturally-occurring(“wild-type”) polynucleic acid or polypeptide. However, in the contextof the present invention a modification of a native isolatedpolynucleotide and polypeptide has occurred to provide a variantpolypeptide with a particular phenotype, for example, amino acidsubstitution in a native glyphosate sensitive EPSPS to provide aglyphosate resistant EPSPS. The polynucleotide modified in this manneris normative with respect to the genetic elements normally found linkedto a naturally occurring unmodified polynucleotide.

Using well-known methods, the skilled artisan can readily producenucleotide and amino acid sequence variants of genes and proteins thatprovide a modified gene product. For example, “variant” DNA molecules ofthe present invention are DNA molecules containing changes in an EPSPScoding sequence, such as, changes that include one or more nucleotidesof a native EPSPS coding sequence being deleted, added, and/orsubstituted, such that the variant EPSPS gene encodes a modified proteinthat retains EPSPS activity and is now resistant to glyphosateherbicide. Variant DNA molecules can be produced, for example, bystandard DNA mutagenesis techniques or by chemically synthesizing thevariant DNA molecule or a portion thereof. Methods for chemicalsynthesis of nucleic acids are discussed, for example, in Beaucage etal., Tetra. Letts. 22:1859-1862 (1981), and Matteucci et al., J. Am.Chem. Soc. 103:3185-(1981). Chemical synthesis of nucleic acids can beperformed, for example, on automated oligonucleotide synthesizers. Suchvariants preferably do not change the reading frame of theprotein-coding region of the nucleic acid. The present invention alsoencompasses fragments of a protein that lacks at least one residue of afull-length protein, but that substantially maintains activity of theprotein.

A first nucleic-acid molecule is “operably linked” with a secondnucleic-acid molecule when the first nucleic-acid molecule is placed ina functional relationship with the second nucleic-acid molecule. Forexample, a promoter is operably linked to a protein-coding nucleic acidsequence if the promoter effects the transcription or expression of thecoding sequence. Generally, operably linked DNA molecules are contiguousand, where necessary to join two protein-coding regions, in readingframe.

The term “plant” encompasses any higher plant and progeny thereof,including monocots (for example, corn, rice, wheat, barley, etc.),dicots (for example, soybean, cotton, canola, tomato, potato,Arabidopsis, tobacco, etc.), gymnosperms (pines, firs, cedars, etc.) andincludes parts of plants, including reproductive units of a plant (forexample, seeds, bulbs, tubers, fruit, flowers, etc.) or other parts ortissues from that the plant can be reproduced.

“Polyadenylation signal” or “polyA signal” refers to a nucleic acidsequence located 3′ to a coding region that causes the addition ofadenylate nucleotides to the 3′ end of the mRNA transcribed from thecoding region.

“Polymerase chain reaction (PCR)” refers to a DNA amplification methodthat uses an enzymatic technique to create multiple copies of onesequence of nucleic acid (amplicon). Copies of a DNA molecule areprepared by shuttling a DNA polymerase between two amplimers. The basisof this amplification method is multiple cycles of temperature changesto denature, then re-anneal amplimers (DNA primer molecules), followedby extension to synthesize new DNA strands in the region located betweenthe flanking amplimers. Nucleic-acid amplification can be accomplishedby any of the various nucleic-acid amplification methods known in theart, including the polymerase chain reaction (PCR). A variety ofamplification methods are known in the art and are described, interalia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in PCR Protocols: AGuide to Methods and Applications, ed. Innis et al., Academic Press, SanDiego, 1990. PCR amplification methods have been developed to amplify upto 22 kb of genomic DNA and up to 42 kb of bacteriophage DNA (Cheng etal., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods aswell as other methods known in the art of DNA amplification may be usedin the practice of the present invention.

The term “promoter” or “promoter region” refers to a polynucleic acidmolecule that functions as a regulatory element, usually found upstream(5′) to a coding sequence, that controls expression of the codingsequence by controlling production of messenger RNA (mRNA) by providingthe recognition site for RNA polymerase and/or other factors necessaryfor start of transcription at the correct site. As contemplated herein,a promoter or promoter region includes variations of promoters derivedby means of ligation to various regulatory sequences, random orcontrolled mutagenesis, and addition or duplication of enhancersequences. The promoter region disclosed herein, and biologicallyfunctional equivalents thereof, are responsible for driving thetranscription of coding sequences under their control when introducedinto a host as part of a suitable recombinant DNA construct, asdemonstrated by its ability to produce mRNA.

A “recombinant” nucleic acid is made by a combination of two otherwiseseparated segments of nucleic acid sequence, for example, by chemicalsynthesis or by the manipulation of isolated segments of polynucleicacids by genetic engineering techniques. The term “recombinant DNAconstruct” refers to any agent such as a plasmid, cosmid, virus,autonomously replicating sequence, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA nucleotide sequence,derived from any source, capable of genomic integration or autonomousreplication, comprising a DNA molecule that one or more DNA sequenceshave been linked in a functionally operative manner. Such recombinantDNA constructs are capable of introducing a 5′ regulatory sequence orpromoter region and a DNA sequence for a selected gene product into acell in such a manner that the DNA sequence is transcribed into afunctional mRNA that is translated and therefore expressed. RecombinantDNA constructs may be constructed to be capable of expressing antisenseRNAs, or stabilized double stranded antisense RNA in order to inhibitexpression of a specific target RNA of interest.

“Resistance” refers to an enzyme that is able to function in thepresence of a toxin, for example, naturally occurring glyphosateresistant class II EPSP synthases resistant to glyphosate or a modifiedEPSPS enzyme having catalytic activity that is unaffected by at aherbicide concentration that normally disrupts the same activity in thewild type enzyme, for example, the modified class I EPSP synthases ofthe present invention. An enzyme that has resistance to a herbicide mayalso have the function of detoxifying the herbicide, for example,phosphinothricin acetyltransferase, and glyphosate oxidoreductase.

“Selectable marker” refers to a polynucleic acid molecule that encodes aprotein, which confers a phenotype facilitating identification of cellscontaining the polynucleic acid molecule. Selectable markers includethose genes that confer resistance to antibiotics (for example,ampicillin, kanamycin), complement a nutritional deficiency (forexample, uracil, histidine, leucine), or impart a visuallydistinguishing characteristic (for example, color changes orfluorescence). Useful dominant selectable marker genes include genesencoding antibiotic resistance genes (for example, neomycinphosphotransferase, npt); and herbicide resistance genes (for example,phosphinothricin acetyltransferase, class II EPSP synthase, modifiedclass I EPSP synthase). A useful strategy for selection of transformantsfor herbicide resistance is described, for example, in Vasil, CellCulture and Somatic Cell Genetics of Plants, Vols. I-III, LaboratoryProcedures and Their Applications Academic Press, New York (1984).

An “artificial polynucleotide” as used in the present invention is a DNAsequence designed according to the methods of the present invention andcreated as an isolated DNA molecule for use in a DNA construct thatprovides expression of a protein in host cells, or for the purposes ofcloning into appropriate constructs or other uses known to those skilledin the art. Computer programs are available for these purposes,including but not limited to the “BestFit” or “Gap” programs of theSequence Analysis Software Package, Genetics Computer Group (GCG), Inc.,University of Wisconsin Biotechnology Center, Madison, Wis. 53711. Theartificial polynucleotide may be created by a one or more methods knownin the art, that include, but are not limited to: overlapping PCR. Anartificial polynucleotide as used herein, is non-naturally occurring andcan be substantially divergent from other polynucleotides that code forthe identical or nearly identical protein.

Expression of a Modified Class I EPSPS Coding Sequence in Plants

DNA constructs are made that contain various genetic elements necessaryfor the expression of the EPSPS coding sequence in plants. “DNAconstruct” refers to the heterologous genetic elements operably linkedto each other making up a recombinant DNA molecule and may compriseelements that provide expression of a DNA polynucleotide molecule in ahost cell and elements that provide maintenance of the construct in thehost cell. A plant expression cassette comprises the operable linkage ofgenetic elements that when transferred into a plant cell providesexpression of a desirable gene product. “Plant expression cassette”refers to chimeric DNA segments comprising the regulatory elements thatare operably linked to provide the expression of a transgene product inplants. Promoters, leaders, introns, transit peptide encodingpolynucleic acids, 3′ transcriptional termination regions are allgenetic elements that may be operably linked by those skilled in the artof plant molecular biology to provide a desirable level of expression orfunctionality to a glyphosate resistant class I EPSPS of the presentinvention. A DNA construct can contain one or more plant expressioncassettes expressing the DNA molecules of the present invention or otherDNA molecules useful in the genetic engineering of crop plants.

A variety of promoters specifically active in vegetative tissues, suchas leaves, stems, roots and tubers, can be used to express the EPSPSpolynucleic acid molecules of the present invention. Examples oftuber-specific promoters include, but are not limited to the class I andII patatin promoters (Bevan et al., EMBO J. 8:1899-1906, 1986;Koster-Topfer et al., Mol Gen Genet. 219:390-396, 1989; Mignery et al.,Gene. 62:27-44, 1988; Jefferson et al., Plant Mol. Biol. 14: 995-1006,1990), the promoter for the potato tuber ADPGPP genes, both the largeand small subunits; the sucrose synthase promoter (Salanoubat andBelliard, Gene. 60:47-56, 1987; Salanoubat and Belliard, Gene 84:181-185, 1989); and the promoter for the major tuber proteins includingthe 22 kd protein complexes and proteinase inhibitors (Hannapel, PlantPhysiol. 101:703-704, 1993). Examples of leaf-specific promotersinclude, but are not limited to the ribulose biphosphate carboxylase(RBCS or RuBISCO) promoters (see, for example, Matsuoka et al., Plant J.6:311-319, 1994); the light harvesting chlorophyll a/b binding proteingene promoter (see, for example, Shiina et al., Plant Physiol.115:477-483, 1997; Casal et al., Plant Physiol. 116:1533-1538, 1998);and the Arabidopsis thaliana myb-related gene promoter (Atmyb5) (Li etal., FEBS Lett. 379:117-121, 1996). Examples of root-specific promoterinclude, but are not limited to the promoter for the acid chitinase gene(Samac et al., Plant Mol. Biol. 25:587-596, 1994); the root specificsubdomains of the CaMV35S promoter that have been identified (Lam etal., Proc. Natl. Acad. Sci. (U.S.A.) 86:7890-7894, 1989); the ORF13promoter from Agrobacterium rhizogenes that exhibits high activity inroots (Hansen et al., Mol. Gen. Genet. 254:337-343 (1997); the promoterfor the tobacco root-specific gene TobRB7 (Yamamoto et al., Plant Cell3:371-382, 1991); and the root cell specific promoters reported byConkling et al. (Conkling et al., Plant Physiol. 93:1203-1211, 1990).

Another class of useful vegetative tissue-specific promoters ismeristematic (root tip and shoot apex) promoters. For example, the“SHOOTMERISTEMLESS” and “SCARECROW” promoters, which are active in thedeveloping shoot or root apical meristems can be used (Di Laurenzio etal., Cell 86:423-433, 1996; Long, Nature 379:66-69, 1996). Anotherexample of a useful promoter is that which controls the expression of3-hydroxy-3-methylglutaryl coenzyme A reductase HMG2 gene, whoseexpression is restricted to meristematic and floral (secretory zone ofthe stigma, mature pollen grains, gynoecium vascular tissue, andfertilized ovules) tissues (see, for example, Enjuto et al., Plant Cell.7:517-527, 1995). Also another example of a useful promoter is thatwhich controls the expression of knI-related genes from maize and otherspecies that show meristem-specific expression (see, for example,Granger et al., Plant Mol. Biol. 31:373-378, 1996; Kerstetter et al.,Plant Cell 6:1877-1887, 1994; Hake et al., Philos. Trans. R. Soc. Lond.B. Biol. Sci. 350:45-51, 1995). Another example of a meristematicpromoter is the Arabidopsis thaliana KNAT1 promoter. In the shoot apex,KNAT1 transcript is localized primarily to the shoot apical meristem;the expression of KNATI in the shoot meristem decreases during thefloral transition and is restricted to the cortex of the inflorescencestem (see, for example, Lincoln et al., Plant Cell 6:1859-1876, 1994).

Suitable seed-specific promoters can be derived from the followinggenes: MAC1 from maize (Sheridan et al., Genetics 142:1009-1020, 1996;Cat3 from maize (GenBank No. L05934, Abler et al., Plant Mol. Biol.22:10131-1038, 1993); viviparous-1 from Arabidopsis (Genbank No.U93215); Atimycl from Arabidopsis (Urao et al., Plant Mol. Biol.32:571-57, 1996; Conceicao et al., Plant 5:493-505, 1994); napA fromBrassica napus (GenBank No. J02798); the napin gene family from Brassicanapus (Sjodahl et al., Planta 197:264-271, 1995, and others (Chen etal., Proc. Natl. Acad. Sci. 83:8560-8564, 1986).

The ovule-specific promoter for BEL1 gene can also be used (Reiser etal. Cell 83:735-742, 1995, GenBank No. U39944; Ray et al, Proc. Natl.Acad. Sci. USA 91:5761-5765, 1994). The egg and central cell specificMEA (FIS1) and FIS2 promoters are also useful reproductivetissue-specific promoters (Luo et al., Proc. Natl. Acad. Sci. USA,97:10637-10642, 2000; Vielle-Calzada, et al., Genes Dev. 13:2971-2982,1999).

A maize pollen-specific promoter has been identified in maize (Guerreroet al., Mol. Gen. Genet. 224:161-168, 1990). Other genes specificallyexpressed in pollen have been described (see, for example, Wakeley etal., Plant Mol. Biol. 37:187-192, 1998; Ficker et al., Mol. Gen. Genet.257:132-142, 1998; Kulikauskas et al., Plant Mol. Biol. 34:809-814,1997; Treacy et al., Plant Mol. Biol. 34:603-611, 1997).

It is recognized that additional promoters that may be utilized aredescribed, for example, in U.S. Pat. Nos. 5,378,619, 5,391,725,5,428,147, 5,447,858, 5,608,144, 5,608,144, 5,614,399, 5,633,441,5,633,435, and 4,633,436. It is further recognized that the exactboundaries of regulatory sequences may not be completely defined, DNAfragments of different lengths may have identical promoter activity.

The translation leader sequence means a DNA molecule located between thepromoter of a gene and the coding sequence. The translation leadersequence is present in the fully processed mRNA upstream of thetranslation start sequence. The translation leader sequence may affectprocessing of the primary transcript to mRNA, mRNA stability ortranslation efficiency. Examples of translation leader sequences includemaize and petunia heat shock protein leaders, plant virus coat proteinleaders, plant rubisco gene leaders among others (Turner and Foster,Molecular Biotechnology 3:225, 1995).

The “3′ non-translated sequences” means DNA sequences located downstreamof a structural polynucleotide sequence and include sequences encodingpolyadenylation and other regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal functions inplants to cause the addition of polyadenylate nucleotides to the 3′ endof the mRNA precursor. The polyadenylation sequence can be derived fromthe natural gene, from a variety of plant genes, or from T-DNA. Anexample of the polyadenylation sequence is the nopaline synthase 3′sequence (nos 3′; Fraley et al., Proc. Natl. Acad. Sci. USA 80:4803-4807, 1983). The use of different 3′ non-translated sequences isexemplified by Ingelbrecht et al., Plant Cell 1:671-680, 1989.

The laboratory procedures in recombinant DNA technology used herein arethose well known and commonly employed in the art. Standard techniquesare used for cloning, DNA and RNA isolation, amplification andpurification. Generally enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like are performedaccording to the manufacturer's specifications. These techniques andvarious other techniques are generally performed according to Sambrooket al. (1989).

The DNA construct of the present invention may be introduced into thegenome of a desired plant host by a variety of conventionaltransformation techniques that are well known to those skilled in theart. “Transformation” refers to a process of introducing an exogenouspolynucleic acid molecule (for example, a DNA construct, a recombinantpolynucleic acid molecule) into a cell or protoplast and that exogenouspolynucleic acid molecule is incorporated into a host cell genome or anorganelle genome (for example, chloroplast or mitochondria) or iscapable of autonomous replication. “Transformed” or “transgenic” refersto a cell, tissue, organ, or organism into which a foreign polynucleicacid, such as a DNA vector or recombinant polynucleic acid molecule. A“transgenic” or “transformed” cell or organism also includes progeny ofthe cell or organism and progeny produced from a breeding programemploying such a “transgenic” plant as a parent in a cross andexhibiting an altered phenotype resulting from the presence of theforeign polynucleic acid molecule.

Methods of transformation of plant cells or tissues include, but are notlimited to Agrobacterium mediated transformation method and theBiolistics or particle-gun mediated transformation method. Suitableplant transformation vectors for the purpose of Agrobacterium mediatedtransformation include those elements derived from a tumor inducing (Ti)plasmid of Agrobacterium tumefaciens, for example, right border (RB)regions and left border (LB) regions, and others disclosed byHerrera-Estrella et al., Nature 303:209 (1983); Bevan, Nucleic AcidsRes. 12:8711-8721 (1984); Klee et al., Bio-Technology 3(7):637-642(1985). In addition to plant transformation vectors derived from the Tior root-inducing (Ri) plasmids of Agrobacterium, alternative methods canbe used to insert the DNA constructs of this invention into plant cells.Such methods may involve, but are not limited to, for example, the useof liposomes, electroporation, chemicals that increase free DNA uptake,free DNA delivery via microprojectile bombardment, and transformationusing viruses or pollen.

DNA constructs can be prepared that incorporate the class I EPSPSvariant coding sequences of the present invention for use in directingthe expression of the sequences directly from the host plant cellplastid. Examples of such constructs suitable for this purpose andmethods that are known in the art and are generally described, forexample, in Svab et al., Proc. Natl. Acad. Sci. USA 87:8526-8530, (1990)and Svab et al., Proc. Natl. Acad. Sci. USA 90:913-917 (1993) and inU.S. Pat. No. 5,693,507. It is contemplated that plastid transformationand expression of the class I EPSPS variants of the present inventionwill provide glyphosate tolerance to the plant cell.

A plasmid expression vector suitable for the introduction of apolynucleic acid encoding a polypeptide of present invention in monocotsusing electroporation or particle-gun mediated transformation iscomposed of the following: a promoter that is constitutive ortissue-specific; an intron that provides a splice site to facilitateexpression of the gene, such as the maize Hsp70 intron (U.S. Pat. No.5,593,874); and a 3′ polyadenylation sequence such as the nopalinesynthase 3′ sequence (T-nos 3′; Fraley et al., Proc. Natl. Acad. Sci.USA 80: 4803-4807, 1983). This expression cassette may be assembled onhigh copy replicons suitable for the production of large quantities ofDNA.

When adequate numbers of cells containing the exogenous polynucleic acidmolecule encoding polypeptides from the present invention are obtained,the cells can be cultured, then regenerated into whole plants.“Regeneration” refers to the process of growing a plant from a plantcell (for example, plant protoplast or explant). Such regenerationtechniques rely on manipulation of certain phytohormones in a tissueculture growth medium, typically relying on a biocide and/or herbicidemarker that has been introduced together with the desired nucleotidesequences. Choice of methodology for the regeneration step is notcritical, with suitable protocols being available for hosts fromLeguminoseae (for example, alfalfa, soybean, clover), Umbelliferae(carrot, celery, parsnip), Cruciferae (for example, cabbage, radish,canola/rapeseed), Cucurbitaceae (for example, melons and cucumber),Gramineae (for example, wheat, barley, rice, maize), Solanaceae (forexample, potato, tobacco, tomato, peppers), various floral crops, suchas sunflower, and nut-bearing trees, such as almonds, cashews, walnuts,and pecans. See, for example, Ammirato et al., Handbook of Plant CellCulture—Crop Species. Macmillan Publ. Co. (1984); Shimamoto et al.,Nature 338:274-276 (1989); Fromm, UCLA Symposium on Molecular Strategiesfor Crop Improvement, Apr. 16-22, 1990. Keystone, Colo. (1990); Vasil etal., Bio/Technology 8:429-434 (1990); Vasil et al., Bio/Technology10:667-674 (1992); Hayashimoto, Plant Physiol. 93:857-863 (1990); andDatta et al., Bio-technology 8:736-740 (1990). Such regenerationtechniques are described generally in Klee et al., Ann. Rev. Plant Phys.38:467-486 (1987).

The development or regeneration of transgenic plants containing theexogenous polynucleic acid molecule that encodes a polypeptide ofinterest is well known in the art. Preferably, the regenerated plantsare self-pollinated to provide homozygous transgenic plants, asdiscussed above. Otherwise, pollen obtained from the regenerated plantsis crossed to seed-grown plants of agronomically important lines.Conversely, pollen from plants of these important lines is used topollinate regenerated plants.

Plants that can be made to have enhanced glyphosate tolerance bypractice of the present invention include, but are not limited to,Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus,avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli,brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava,cauliflower, celery, cherry, cilantro, citrus, clementines, coffee,corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole,eucalyptus, fennel, figs, forest trees, gourd, grape, grapefruit, honeydew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine,mango, melon, mushroom, nut, oat, okra, onion, orange, an ornamentalplant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon,pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin,quince, radiata pine, radicchio, radish, raspberry, rice, rye, sorghum,Southern pine, soybean, spinach, squash, strawberry, sugarbeet,sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco,tomato, turf, a vine, watermelon, wheat, yams, and zucchini.

The following examples are provided to better elucidate the practice ofthe present invention and should not be interpreted in any way to limitthe scope of the present invention. Those skilled in the art willrecognize that various modifications, additions, substitutions,truncations, etc., can be made to the methods and genes described hereinwhile not departing from the spirit and scope of the present invention.

EXAMPLES Example 1 Site-Directed Mutagenesis of a Class I EPSPS

Mutagenesis of a DNA molecule encoding a class I EPSPS was directed at aregion of the protein defined by a polypeptide sequence -G-T-X₁-X₂-R-P-(SEQ ID NO:1) of the class I EPSPS, where X₁ and X₂ are any amino acid.The invention described herein provides for the mutagenesis of a geneencoding a class I EPSPS, wherein the mutagenesis results in apolypeptide sequence of -G-X₄-X₁-X₂-R-X₃- (SEQ ID NO:2) in this regionof the class I EPSPS protein related to the binding of the enzymesubstrate and the glyphosate molecule. The amino acid substitutions inSEQ ID NO:1 that will result in a glyphosate resistant class I EPSPSinclude replacing the native threonine (T) at X₄ with amino acidsisoleucine (I) or leucine (L), and replacing the native proline (P) atX₃ with threonine, glycine, cysteine, alanine, or isoleucine. The aminoacid positions 102 and 106 are designated according to the maize EPSPSpolypeptide sequence shown in FIG. 1, however, other plant class I EPSPScoding sequences (FIG. 2), for example, petunia and soybean can be usedas templates for site-directed mutagenesis as the relative positions ofthe threonine and proline amino acids, respectively, are conserved;however, a slightly different amino acid position number in the EPSPSpolypeptide sequence may occur because of variations in the startingpoint of mature EPSPSs from various sources (U.S. Pat. No. 5,866,775,FIG. 1), those variations are recognized by those skilled in the art andare within the scope of the present invention. In a similar manner,site-directed mutagenesis of prokaryote class I EPSPS DNA codingsequences, for example, E. coli (FIG. 2) can be performed usingmutagenesis primers designed to hybridize to these DNA molecules tocreate the EPSPS variants -G-X₄-X₁-X₂-R-X₃- (SEQ ID NO:2) as describedherein.

Mutations were made using plant EPSPS DNA coding sequence template as anexample of class I EPSPSs. Mutations of the DNA coding sequence resultin variant EPSPS protein molecules by the substitution of codons(Table 1) encoding for amino acids in the DNA sequence. The variantprotein sequences that have two amino acid substitutions compared to thewild type protein sequence are referred to as double variants, a singleamino acid substitution is referred to as a single variant. All thevariants were made using the PCR-based QuickChange™ Site-directedMutagenesis Kit (Stratagene, La Jolla, Calif., Cat. No. 200518)following the manufactures instructions. The DNA sequence of eachmutagenesis primer was designed, and then ordered from Invitrogen Corp.,Custom Primers (Carlsbad, Calif.). Mutagenesis of a maize wild-type DNAmolecule (SEQ ID NO:3) encoding the EPSPS enzyme was performed usingpMON70461 (FIG. 3) as the template. pMON70461 contains the unmodifiedwild-type maize EPSPS coding sequence. The previously known T102I, P106Svariant (TIPS) was created by the PCR mediated mutagenesis method usingprimer pairs, TIPSMut-1-U (SEQ ID NO:4) and TIPSMut-2-L (SEQ ID NO:5) asshown in FIG. 4A, and is contained in pMON70462 plasmid. The singleEPSPS variants were created by mutagenesis of the maize wild-type EPSPSDNA coding sequence as controls for measuring the efficacy of the doublevariant EPSPSs. The following single EPSPS variants were created usingPCR mutagenesis: the T102I variant (primers I1-U (SEQ ID NO:6) and I2-L(SEQ ID NO:7), pMON58455), the P106T variant (primers mEmut-9-U (SEQ IDNO:8) and mEmut-10-L (SEQ ID NO:9), pMON70467, FIG. 9), the P106Svariant (primers mEmut-7-U (SEQ ID NO:10) and mEmut-8-L (SEQ ID NO:11),pMON70466), and the P106L variant (primers H1-U (SEQ ID NO:12) and H2-L(SEQ ID NO:13), pMON58451) were created using the unmodified wild-typemaize EPSPS coding sequence contained in pMON70461 as the template forsite directed mutagenesis.

The double variants of the present invention were made using pMON58452(FIG. 5) as the template. This pMON58452 EPSPS gene template containsthe maize EPSPS double variant T102I, P106T (TIPT) that was constructedby mutagenesis of pMON70467 with the mutagenesis primers mEmut-9-U andmEmut-10-L. The various mutagenesis primer sequences were designed andthen were synthesized by Invitrogen Corp., Custom Primers and used incombination in a PCR to create the variant EPSPS coding sequences. ThePCR was set up in a 50 μl reaction in the following manner: dH₂O 38 μl;2 mM dNTP 1 μL; 10× buffer 5 μL; pMON58452 1 μL (10 ng); primer-U 2 μL;primer-L 2 μL; pfu Turbo enzyme 1 μL. PCR was carried out on a MJResearch PTC-200 thermal cycler using the following program: Step 1—94°C. for 30 seconds; Step 2—94° C. for 30 seconds; Step 3—55° C. for 1minute; Step 4—68° C. for 14 minute; Step 5—go to step 2, 16 times; Step6—End. At the end of the PCR, 1 μl of the restriction enzyme DpnI wasadded to each 50 μl of PCR reaction and the mixture was incubated at 37°C. for 1 hour. The proline (106) amino acid codon of pMON58452 wassubstituted in subsequent steps to provide additional variants thatinclude TWO (primer P106G-U, SEQ ID NO:14 and -L, SEQ ID NO:15), TIPA(primer P106A-U, SEQ ID NO:16 and -L, SEQ ID NO:17), TIPV (primerP106V-U, SEQ ID NO:18 and -L, SEQ ID NO:19), TIPL (primer P106L-U, SEQID NO:20 and -L, SEQ ID NO:21), TIPI (primer P106I-U, SEQ ID NO:22 and-L, SEQ ID NO:23), TIPM (primer P106M-U, SEQ ID NO:24 and -L, SEQ IDNO:25), and TIPC (primer P106C-U, SEQ ID NO:26 and -L, SEQ ID NO:27).The mutagenesis primers' DNA sequences are shown in FIGS. 4A and 4B. Thedouble variants of the gene encoding the EPSPS protein were generatedusing the PCR conditions described above.

At the end of the PCR, 1 μl of the restriction enzyme DpnI was added toeach 50 μl of PCR reaction and the mixture was incubated at 37° C. for 1hour. DpnI is a methylation- and hemimethylation-specific restrictionenzyme and will cleave only those double-stranded DNA plasmid containingat least one wild-type, methylated, strand, leaving the mutated plasmidintact. After the DpnI treatment, 1 μl of the treated reaction mixturewas used to transform the competent E. coli strain XL1-blue (StratageneCorp, La Jolla, Calif.) following the manufacturer's instruction. Thetransformed cells were plated onto a Petri dish containing carbenicillinat a final concentration of 0.1 mg/mL. The dish was then incubated at37° C. overnight. Single colonies were picked the next day and used toinoculate a 3 mL liquid culture containing 0.1 mg/mL carbenicillin. Theliquid culture was incubated overnight at 37° C. with agitation at 250rpm. Plasmid DNA was prepared from 1 mL of the liquid culture usingQiagen's miniprep Kit (Qiagen Corp. Cat. No. 27160). The DNA was elutedin 50 μl of dH₂O. The entire coding region of three independent clonesfrom each mutagenesis was sequenced by DNA sequence analysis (ABI Prism™377, PE Biosystems, Foster City, Calif. and DNASTAR sequence analysissoftware, DNASTAR Inc., Madison, Wis.) and confirmed to contain thedesired mutation.

Other plant class I EPSPS coding sequences were modified to contain theTIPA variant. The EPSPS coding sequence of Arabidopsis thaliana(Columbia) EPSPS1 and EPSPS coding sequences of lettuce (Lactuca sativa)were isolated for mutagensis. RT-PCT was used to isolate the codingsequence of the mature protein of both AtEPSPS1 and lettuce EPSPS. Allof the primers were ordered from Invitrogen. The leaf tissues of bothArabidopsis and lettuce were ground into powder in liquid nitrogen witha mortar and pestle. Total RNA was isolated using Qiagene's RNeasy minikit (cat. #74904) using 10 mg leaf powder and RNA was eluted into 50 μlwater. RT-PCR reactions were performed using one-step-RT-PCR kit(Invitrogen #10928-034) in a 50 μL reaction containing: dH₂O 24 μL;reaction buffer 50 μL; total RNA 20 μL; AtEPSPS-F primer (SEQ ID NO:28)(10 μM) 1 μL; AtEPSPS-R (SEQ ID NO:29) (10 μM) 1 μL; Taq 1 μL. RT-PCRwas carried out on a MJ Research PTC-200 thermal cycler using thefollowing program: Step 1—40° C. for 30 seconds; Step 2—94° C. for 2minutes; Step 3—94° C. for 20 seconds; Step 4—65° C. for 30 seconds;Step 5—68° C. for 1 minute 30 seconds; Step 6—go to step 3, 30 times;Step 7—End. Both PCR reactions yielded an approximately 1.3 kilo baseband on a 1 percent agarose electrophoresis gel. The lettuce EPSPScoding sequence was isolated using the primers LsEPSPS-F (SEQ ID NO:30)and LsEPSPS-R (SEQ ID NO:31) in the above described method. The RT-PCRproducts were cloned into PCR-II vector (Invitrogen Corp.) and the DNAmolecules sequenced.

The Arabidopsis and lettuce EPSPS TIPA variants were generated using thePCR-based QuickChange™ Site-directed Mutagenesis Kit (Stratagene Cat.No. 200518) and the DNA mutagenesis primers AtEPSPS-TIPA-F (SEQ IDNO:32) and AtEPSPS-TIPA-R (SEQ ID NO:33) for the Arabidopsis EPSPScoding sequence mutation, and LsEPSPS-TIPA-F (SEQ ID NO:34) andLsEPSPS-TIPA-R (SEQ ID NO:35) for the lettuce EPSPS coding sequencemutation and the PCR conditions were as described above. Two additionalsite-directed mutagenesis reactions were performed to engineer arestriction enzyme site, Nde1, at the 5′ end and to remove an internalNde1 site in the lettuce EPSPS coding sequence. The DNA fragments of theArabidopsis and the lettuce EPSPS TIPA variants were then digested withNde1 and Xho1 and cloned into a pET19 vector. The DNA molecules thatencode for the variant Arabidopsis EPSPS and variant lettuce EPSPS areshown in SEQ ID NO:36 and SEQ ID NO:37, respectively. Examples of plantand bacterial expression DNA constructs that were made with thesevariant EPSPS coding sequences are illustrated in pMON81548 (LsTIPAvariant, promoter of U.S. Pat. No. 6,660,911) shown in FIG. 11 andpMON58491 (AtTIPA variant) shown in FIG. 12. Any of the variant EPSPScoding sequences of the present invention can be inserted into a plantexpression cassette, for example, that contained in pMON81519 orpMON81548 by replacing the existing coding sequence.

Example 2 EPSPS Purification and Enzyme Analysis

The wild type and variant EPSPS coding sequences were cloned into apET-19b base vector (Novagen, Madison, Wis.). The plant (maize) class IEPSPS variants so created were assigned pMON plasmid numbers (Table 2).The variant EPSPS proteins were purified from the E. coli host using theprotocols outlined in the pET system manual, 9th edition (Novagen) or bythe following method. A single colony or a few microliters from aglycerol stock was inoculated into 4 mL LB medium containing 0.1 mg/mLcarbenicillin antibiotic. The culture was incubated with shaking at 37 Cfor 4 hours. The cultures were stored at 4° C. overnight. The followingmorning, 1 mL of the overnight culture was used to inoculate 100 mL offresh LB medium containing 0.1 mg/mL carbenicillin. The cultures wereincubated with shaking at 37 C for 4-5 hours, then the cultures wereplaced at 4° C. for 5-10 minutes. The cultures were then induced withIPTG (1 mM final concentration) and incubated with shaking at 30 C for 4hours or 20° C. overnight. The cells were harvested by centrifugation at7000 rpm for 20 minutes at 4° C. The supernatant was removed and thecells were frozen at −70° C. until further use. The proteins wereextracted by resuspending the cell pellet in BugBuster reagent (Novagen)using 5 mL reagent per gram of cells. Benzonase (125 Units) was added tothe resuspension and the cell suspension was then incubated on arotating mixer for 20 minutes at room temperature. The cell debris wasremoved by centrifugation at 10,000 rpm for 20 minutes at roomtemperature. The supernatant was passed through a 0.45 μm syringe-endfilter and transferred to a fresh tube. A pre-packed columns containing1.25 mL of His-Bind resin was equilibrated with 10 mL of 5 mM imidazole,0.5 M NaCl, 20 mM Tris-HCl pH 7.9 (1× Binding Buffer). The column wasthen loaded with the prepared cell extract. After the cell extract haddrained, the column was then washed with 10 mL of 1× Binding Buffer,followed with 10 mL of 60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl pH7.9 (1× Wash Buffer). The protein was eluted with 5 mL of 1 M imidazole,0.5 M NaCl, 20 mM Tris-HCl pH 7.9 (1× elution buffer). Finally theprotein was dialyzed into 50 mM Tris-HCl pH 6.8. The resulting proteinsolution was concentrated to ˜0.1-0.4 mL using Ultrafree—centrifugaldevice (Biomax-10K MW cutoff, Millipore Corp, MA). Proteins were dilutedto 10 mg/mL and 1 mg/mL in 50 mM Tris pH 6.8, 30 percent final glyceroland stored at −20° C. Protein concentration was determined using Bio-Radprotein assay (Bio-Rad Laboratories, CA). Bovine serum albumin was usedto generate a standard curve 1-5 μg. Samples (10 μL) were added to wellsin a 96 well-plate and mixed with 200 μL of Bio-Rad protein assayreagent (1 part dye reagent concentrate: 4 parts water). The sampleswere read at OD₅₉₅ after ˜5 minutes using a spectraMAX 250 plate reader(Molecular Devices Corporation, Sunnyvale, Calif.) and compared to thestandard curve.

The EPSPS enzyme assays contained 50 mM K⁺-HEPES pH 7.0 and 1 mMshikimate-3-phosphate (Assay mix). The K_(m)-PEP were determined byincubating assay mix (30 μL) with enzyme (10 μL) and varyingconcentrations of [¹⁴C] PEP in a total volume of 50 μL. The reactionswere quenched after various times with 50 μL of 90 percent ethanol/0.1 Macetic acid pH 4.5 (quench solution). The samples were centrifuged at14,000 revolutions per minute and the resulting supernatants wereanalyzed for ¹⁴C-EPSP production by HPLC. The percent conversion of¹⁴C-PEP to ¹⁴C-EPSP was determined by HPLC radioassay using an AX100weak anion exchange HPLC column (4.6×250 mm, SynChropak) with 0.26 Misocratic potassium phosphate eluant, pH 6.5 at 1 mL/minute mixed withUltima-Flo AP cocktail at 3 mL/min (Packard). Initial velocities werecalculated by multiplying fractional turnover per unit time by theinitial concentration of the substrate.

The inhibition constant (K_(i)) were determined by incubating assay mix(30 μL) with and without glyphosate and ¹⁴C-PEP (10 μL of 2.6 mM). Thereaction was initiated by the addition of enzyme (10 μL). The assay wasquenched after 2 minutes with quench solution. The samples werecentrifuged at 14,000 rpm and the conversion of ¹⁴C-PEP to ¹⁴C-EPSP wasdetermined as shown above. The steady-state and IC₅₀ data were analyzedusing the GraFit software (Erithacus Software, UK). The K_(i) valueswere calculated from the IC₅₀ values using the following algorithm:K_(i)=[IC]₅₀/(1+[S]/K_(m)). The assays were done such that the ¹⁴C-PEPto ¹⁴C-EPSP turnover was ≦30 percent. In these assays bovine serumalbumin (BSA) and phosphoenolpyruvate (PEP) were obtained from Sigma.Phosphoenol-[1-¹⁴C]pyruvate (29 mCi/mmol) was from Amersham Corp.(Piscataway, N.J.).

The maize EPSPS variants that were cloned into pET-19b and from whichproteins were expressed and assayed, included the double variants TIPS,TIPT, TIPG, TIPC, TIPA, TIPV, TIPM, TIPL, and TIPI; and single variantsT102I, P106S, P106T, and P106L (Table 2). The enzymes were purified andassayed for apparent K_(m) of PEP (K_(m)-PEP) and inhibition byglyphosate (K_(i)). The TIPS variant is well known and is currently inthe commercial Roundup Ready® corn product GA21 (U.S. Pat. No.6,040,497) and its kinetic parameters serve as the baseline value for aglyphosate resistant class I EPSPS enzyme that is sufficient to provideglyphosate tolerance to a transgenic plant. All the variants werecharacterized and the kinetic parameters are shown in Table 2.Substantial differences were observed between these variants.Surprisingly, the results showed that two of the new variants, TIM andTIPT, were more resistant to glyphosate than the TIPS variant anddemonstrated a similar K_(m)-PEP. These EPSPS enzyme double variantswill provide enhanced glyphosate tolerance when appropriately expressedin transgenic plants. The variant TIPG has similar K_(m)-PEP as thewild-type enzyme (WT), but has a K_(i) of only 38.6 μM, not animprovement over TIPS, but this K_(i) should be sufficient to provideglyphosate tolerance in transgenic plants when appropriately expressed.The variants TIPC and TIPI show a high level of resistance to glyphosatebut have 1.7-fold and 2.2-fold higher K_(m)-PEP than the wild-typeenzyme, respectively. Although TIPC and TIPI are somewhat less efficientthan the wild-type enzyme for K_(m)-PEP, they do show a high level ofresistance to glyphosate and when these are overexpressed as a transgenein plant cells, these enzymes should be sufficient to provide glyphosatetolerance. Other double variants that include TIPV, TIPM, TIPL showedvery high resistance to glyphosate, but have significantly higher K_(m)for PEP and therefore do not have sufficient substrate binding activityto provide useful EPSPS enzyme activity to a transgenic plant.Additional double variants TIPD and TIPN had a K_(m) for PEP of 355 and566, respectively and were not assayed for K_(i) for glyphosate becausethe substrate binding activity was too inefficient for these variants tohave effective EPSPS activity. For comparison purposes, the enzymekinetics of the naturally occurring class II glyphosate resistant EPSPSisolated from Agrobacterium strain CP4 (CP4 EPSPS) was expressed frompMON21104 (RecA promoter/G10 leader/CP4 EPSPS/T7 terminator) and assayedunder the same conditions as the maize variant EPSPSs and demonstrated aK_(m)-PEP of 14.4 μM and K_(i) for glyphosate of 5100 μM.

TABLE 2 Steady-state kinetic parameters of maize EPSPS variants pMON#EPSPS variant K_(m)-PEP (μM) K_(i)-glyp (μM) pMON70461 WT 27 ± 4  0.50 ±0.06 PMON70462 TIPS 10.6 ± 1.6 58.0 ± 14  PMON58452 TIPT 11.2 ± 1.8101.3 ± 12.7 PMON42480 TIPG 23.0 ± 3.7 38.6 ± 1.7 PMON42485 TIPC 47.0 ±4.0 818.2 ± 74.4 PMON42481 TIPA 10.2 ± 1.1 148.3 ± 18.3 PMON42486 TIPI60.3 ± 2.8 2500 ± 900 pMON42482 TIPV 109.3 ± 12.9 1600 ± 400 pMON42484TIPM 143.3 ± 12.6 37200 ± 1500 pMON42483 TIPL 99.5 ± 8.9 2100 ± 100pMON58455 T102I 233.0 ± 25.5 148.6 ± 12.4 pMON70466 P106S 17.1 ± 2.8 1.0 ± 0.1 pMON70467 P106T 24.6 ± 4.4  4.0 ± 0.6 pMON58451 P106L 86.7 ±5.7 28.6 ± 0.1 pMON21104 CP4 EPSPS 14.4 ± 2.4 5100 ± 0.1 

Example 3

The maize EPSPS double variants ZmTIPT (SEQ ID NO:38) and ZmTIPA (SEQ IDNO:39) were made with as a CTP translational fusion into plantexpression DNA constructs, pMON70472 (FIG. 7) and pMON70475 (FIG. 8),respectively. pMON30167 (FIG. 6) was digested with SphI/NotI and EcoRI,a Not I/Eco RI backbone DNA fragment of pMON30167 and a Sph I/Not I DNAfragment (rice actin promoter, P-OsAct1 and intron, I-Os.Act1, U.S. Pat.No. 5,641,876) were gel purified. A maize EPSPS-TIPT DNA molecule wasisolated from pMON58452 by incorporating Sph I and Eco RI endonucleasesites in the ends with DNA primer molecules ZmAroA-1 (SEQ ID NO:40) andZmAroA-2 (SEQ ID NO:41). The amplified mEPSPS-TIPT DNA fragment wasdigested with SphI and Eco RI and gel purified. A triple ligation wasperformed with the two pMON30167 fragments and the modified maizeEPSPS-TIPT DNA fragment. The ligated plasmid was transformed into E.coli strain XL1-blue following the manufacturer's instruction andscreened for colonies with the correct plasmid. The mature maize EPSPSN-terminus was restored (should be Ala not Met) by mutagenesis withStratagene QuikChange kit according to manufacturer's instruction andusing DNA primer molecules ZmAroA-3 (SEQ ID NO:42) and ZmAroA-4 (SEQ IDNO:43) contained in pMON70472 (FIG. 7).

Example 4

A DNA construct containing the TIPT variant (pMON70472, FIG. 7) underthe control of rice actin promoter was transformed into corn plant cells(LH198 x Hill) by an Agrobacterium mediated transformation method. Forexample, a disarmed Agrobacterium strain C58 harboring the binary DNAconstruct of the present invention is used. The DNA construct istransferred into Agrobacterium by a triparental mating method (Ditta etal., Proc. Natl. Acad. Sci. 77:7347-7351, 1980). Liquid cultures ofAgrobacterium are initiated from glycerol stocks or from a freshlystreaked plate and grown overnight at 26° C.-28° C. with shaking(approximately 150 rpm) to mid-log growth phase in liquid LB medium, pH7.0 containing the appropriate antibiotics. The Agrobacterium cells areresuspended in the inoculation medium (liquid CM4C) and the density isadjusted to OD₆₆₀ of 1. Freshly isolated Type II immature HiIIxLH198 andHiII corn embryos are inoculated with Agrobacterium containing aconstruct and co-cultured several days in the dark at 23° C. The embryosare then transferred to delay media and incubated at 28° C. for severalor more days. All subsequent cultures are kept at this temperature. Theembryos are transferred to a first selection medium containingcarbenicillin 500/0.5 mM glyphosate). Two weeks later, surviving tissueare transferred to a second selection medium containing carbenicillin500/1.0 mM glyphosate). Subculture surviving callus every 2 weeks untilevents can be identified. This may take about 3 subcultures on 1.0 mMglyphosate. Once events are identified, bulk up the tissue toregenerate. The plantlets (events) are transferred to MSOD media inculture vessel and kept for two weeks. The transformation efficiency isdetermined by dividing the number of events produced by the number ofembryos inoculated. Then the plants with roots are transferred intosoil. Those skilled in the art of monocot transformation methods canmodify this method to provide substantially identical transgenic monocotplants containing the DNA compositions of the present invention, or useother methods, such as, particle gun, that are known to providetransgenic monocot plants.

The results of molecular analysis and glyphosate selection of theregenerated corn events transformed with a DNA construct containingZmTIPT are shown in Table 3. The events were analyzed for single copyinsertion in the corn genome and for vegetative and male fertility.Eight of the thirteen transgenic events (61%) containing the pMON70472(TIPT) plant expression cassette were assayed using Taqman® analysis(ABI, Foster City, Calif.) and determined to be a single copy insertinto the corn genome. The events were treated with a foliar applicationof 32 oz/acre of Roundup® Ultra at the V4 stage of corn development andthen another foliar application of 64 oz/acre Roundup® Ultra at aboutV7. The treated plants were scored for vegetative (glypT) andreproductive tolerance (fertile) to glyphosate. Four of the eight singlecopy events (50%) showed no vegetative injury due to the glyphosateapplication at V4 (% V4 glypT). A 64 oz Roundup® Ultra treatment wasapplied at around the V7 stage and the plants were scored for vegetativeglyphosate tolerance and male fertility (% V7glypT/fertile). All four ofthe events 100% (4/4) that were single copy and vegetatively tolerant toglyphosate at V4 were also vegetatively tolerant and fully male fertileafter the V7 treatment. Compared to the commercial standard glyphosateresistant EPSPS(CP4 EPSPS) the ZmTIPT variant performed surprisinglywell. DNA constructs containing the TIPT variant of a class I EPSPSprovide plants that are vegetatively and reproductively tolerant to aglyphosate containing herbicide.

TABLE 3 Results of glyphosate tolerance treatment of corn eventscontaining the TIPT EPSPS variant. DNA Construct #events single copy %V4 glypT % V7 glypT/fertile pMON70472 (ZmTIPT) 13 61% (8/13) 50% (4/8)100% (4/4) pMON30167 (CP4 EPSPS) 24 33% (8/24) 62% (5/8) 100% (5/5)

Example 5

The ZmTIPT variant was constructed into a plant expression constructsuitable for dicot plant expression. The DNA construct was designatedpMON81519 and is illustrated in FIG. 10. The DNA construct and controlconstructs were transferred into Agrobacterium by a triparental matingmethod as previously described. The transformed Agrobacterium cells wereused to transfer the plant expression cassette into Arabidopsis andtobacco cells.

Arabidopsis embryos were transformed by an Agrobacterium mediated methodessentially as described by Bechtold N, et al., CR Acad Sci ParisSciences di la vie/life sciences 316: 1194-1199, (1993). AnAgrobacterium strain ABI containing a DNA construct is prepared asinoculum by growing in a culture tube containing 10 mls Luria Broth andantibiotics. The Agrobacterium inoculum is pelleted by centrifugationand resuspended in 25 ml Infiltration Medium (MS Basal Salts 0.5%,Gamborg's B-5 Vitamins 1%, Sucrose 5%, MES 0.5 g/L, pH 5.7) with 0.44 nMbenzylaminopurine (10 ul of a 1.0 mg/L stock in DMSO per liter) and0.02% Silwet L-77 to an OD₆₀₀ of 0.6.

Mature flowering Arabidopsis plants are vacuum infiltrated in a vacuumchamber with the Agrobacterium inoculum by inverting the pots containingthe plants into the inoculum. The chamber is sealed, a vacuum is appliedfor several minutes, release the vacuum suddenly, blot the pots toremove excess inoculum, cover pots with plastic domes and place pots ina growth chamber at 21° C. 16 hours light and 70% humidity.Approximately 2 weeks after vacuum infiltration of the inoculum, covereach plant with a Lawson 511 pollination bag. Approximately 4 weeks postinfiltration, withhold water from the plants to permit dry down. Harvestseed approximately 2 weeks after dry down.

The transgenic Arabidopsis plants produced by the infiltrated seedembryos are selected from the nontransgenic plants by a germinationselection method. The harvested seed is surface sterilized then spreadonto the surface of selection media plates containing MS Basal Salts 4.3g/L, Gamborg's B-5 (500×) 2.0 g/L, MES 0.5 g/L, and 8 g/L Phytagar withCarbenicillin 250 mg/L, Cefotaxime 100 mg/L, and PPM 2 ml/L and 300 μMglyphosate added as a filter sterilized liquid solution, afterautoclaving. The pMON81519 V1 events and control construct pMON81517glyphosate tolerant transgenic Arabidopsis plants are selected by sprayapplication of glyphosate herbicide at a rate of 24 ounces/acre, thesurviving plants are transplanted into individual pots. The V1 plantsare sprayed a second time corresponding to the observation of bolting,approximately 16 days after the at a rate of 24 ounces/acre. The secondspray will determine the efficacy of the two constructs for conferringreproductive tolerance. The plants are observed for vegetative andreproductive effects of glyphosate application. Sixty-two plants wereassayed that were transformed with the control construct pMON81517 thatcontains the CP4 EPSPS (class II EPSPS) coding sequence, forty-nineplants were assayed that were transformed with pMON81519. The resultsshown in Table 4 demonstrate that the percentage of plants showingglyphosate tolerance and fertility is about the same for the ZmTIPTclass I EPSPS variant as for the class II EPSPS.

Tobacco is a well known model plant for testing of transgene constructsand the methods of transformation are well known in the art of planttransformation. Briefly, tobacco leaf tissue is cut and placed ontosolid pre-culture plates containing the appropriate culture medium. Theday before Agrobacterium inoculation, a 10 μl loop of a transformedAgrobacterium culture containing pMON81519 or control construct isplaced into a tube containing 10 mls of YEP media with appropriateantibiotics to maintain selection of the DNA construct. The tube is putinto a shaker to grow overnight at 28° C. The OD₆₀₀ of the Agrobacteriumis adjusted to 0.15-0.30 OD₆₀₀ with TXD medium. Inoculate tobacco leaftissue explants by pipetting 7-8 mls of the liquid Agrobacteriumsuspension directly onto the pre-culture plates covering the explanttissue. Allow the Agrobacterium to remain on the plate for 15 minutes.Tilt the plates and aspirate liquid off using a sterile 10 ml wide borepipette. The explants are co-cultured on these same plates for 2-3 days.The explants are then transferred to fresh medium containing appropriateselection agents and maintained for 3-4 weeks at which time the callustissue is transferred to fresh medium. At 6-8 weeks, shoots should beexcised from the callus allowed to root in culture media. Rooted shootsare then transferred to soil after 2-3 weeks. The plants are treatedwith 16-24 oz/Acre glyphosate and scored for vegetative and reproductivetolerance. The results shown in Table 4 demonstrate that the percentageof plants showing glyphosate tolerance and fertility is about the samefor the ZmTIPT class I EPSPS variant as for the class II EPSPS.

TABLE 4 Results of glyphosate tolerance treatment of Arabidopsis andtobacco events containing the TIPT EPSPS variant. Tobacco ArabidopsisDNA Construct % glypT/fertile % glypT/fertile PMON81517 (CP4 EPSPS) 56%(N = 41) 61% (N = 62) PMON81519 (ZmTIPT) 49% (N = 39) 65% (N = 49)

Example 6

Class I EPSPSs can be modified by site-directed mutagenesis methods orrandom mutagenesis method to provide an enzyme that is resistant toglyphosate. The present invention preferably provides amino acidsubstitutions of the Thr102 and Pro106 positions. In addition to thepreviously described TIP-T,G,C,A, and I variants of the presentinvention, an additional substitution was performed of the Thr102 codonwas replaced with a leucine (L, Leu) codon and the Pro106 codon wasreplaced with an alanine (A, Ala) codon by site-directed modification ofthe corresponding codons in a maize EPSPS DNA coding sequence resultingin a variant ZmTLPA (SEQ ID NO:44) that provides a glyphosate resistantenzyme. In another variant, the Thr102 codon was replaced with a codonfor Glutamine (Q, Gln), the Pro106 codon modified to an Ala codon,resulting in a TQPA variant.

These maize EPSPS variants, TLPA and TQPA were generated using thePCR-based QuickChange™ Site-directed Mutagenesis Kit by Stratagene (Cat.No. 200518). The unmodified maize EPSPS coding sequence was used as thetemplate for PCR to generate the variants. The mutagenesis oligo primerswere ordered from Invitrogen. The PCR was set up in a 50 μl reaction inthe following manner: dH₂O 38 μl; 2 mM dNTP 1 μL; 10× buffer 5 μL;pMON70461 1 μL (10 ng); ZmTLPA-1 (SEQ ID NO:45) 2 μL; ZmTLPA-2 (SEQ IDNO:46) 2 μL; pfu Turbo enzyme 1 μL. The PCR was carried out on a MJResearch PTC-200 thermal cycler using the following program: Step 1—94°C. for 2′; Step 2—94° C. for 30″; Step 3—55° C. for 30″; Step 4—68° C.for 14′; Step 5—go to step 2, 16 times; Step 6—End. At the end of thePCR, 1 μl of the restriction enzyme DpnI was added to each 50 μl of PCRreaction and the mixture was incubated at 37° C. for 1 hour. After theDpnI treatment, 1 μl of the treated reaction mixture was used totransform the competent E. coli strain XL1-blue strain (Stratagene)following the manufacturer's instruction. The transformed cells wereplated on a Petri dish containing carbenicillin at a final concentrationof 0.1 mg/mL. The plate was then incubated at 37° C. overnight. Singlecolonies were picked the next day and used to inoculate a 3 mL liquidculture containing 0.1 mg/mL carbenicillin. The liquid culture wasincubated overnight at 37° C. with agitation at 250 rpm. Plasmid DNA wasprepared from 1 mL of the liquid culture using Qiagen's miniprep Kit(Cat. No. 27160). The DNA was eluted in 50 μl of dH₂O. The entire codingregion of three independent clones from each mutagenesis was sequencedand confirmed to contain the desired mutation. The variant codingsequences were inserted into a pET19 expression vector in the properorientation to provide expression of the variant enzyme as atranslational fusion with a purification tag.

The mutant maize EPSPS enzymes (TLPA and TQPA) were assayed forcatalytic activity, substrate binding, and resistance to glyphosate(K_(i)) using the assay conditions previously described. The results areshown in Table 5. These mutants were compared to the wild type (WT)unmodified maize EPSPS, and the TIPA variant. The results provideevidence that the TLPA variant is resistant to glyphosate and hassufficient enzyme kinetics that when expressed in a transgenic plantwill provide glyphosate tolerance to the transgenic plant when fusedwith a CTP or modified for chloroplast expression. Further amino acidsubstitutions at the 106 position that include threonine, glycine,cysteine and isoleucine are expected to result in a glyphosate resistantenzyme as observed in combination with the T-I modification at position102.

TABLE 5 EPSPS steady-state kinetics k_(cat)/K_(m) Enzyme k_(cat) (s⁻¹)K_(m)-PEP (□M) (□M⁻¹s⁻¹) Ki (□M) WT maize 8.8 ± 0.5 27 ± 4 0.3  0.5 ±0.06 TIPA 2.1 ± 0.1 10.2 ± 1.1 0.2 148.3 ± 18.3 TLPA 2.4 ± 0.1 13.1 ±2.5 0.2 46.8 ± 7.6 TQPA 3.8 ± 0.2 163.8 ± 22.9 0.02 2200 ± 200

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1. An isolated DNA molecule that encodes a glyphosate resistant class IEPSPS protein, wherein said glyphosate resistant class I EPSPS proteincomprises a polypeptide sequence GX₄X₁X₂RX₃, where X₁ and X₂ are anyamino acid, X₄ is isoleucine or leucine, and X₃ is selected from thegroup consisting of threonine, glycine, cysteine, alanine, andisoleucine.
 2. The DNA molecule of claim 1, wherein said DNA molecule isderived from a plant genome.
 3. The DNA molecule of claim 2, whereinsaid DNA molecule is derived from Zea mays and is modified to encode fora glyphosate resistant class I EPSPS protein comprising an isoleucine atposition 102 and an amino acid at position 106 selected from the groupselected from threonine, glycine, cysteine, alanine and isoleucine. 4.The DNA molecule of claim 2, wherein said DNA molecule is derived fromArabidopsis and is modified to encode for said glyphosate resistantclass I EPSPS protein comprising an isoleucine at position 102 and analanine at position
 106. 5. The DNA molecule of claim 2, wherein saidDNA molecule is derived from Lettuce and is modified to encode for saidglyphosate resistant class I EPSPS protein comprising an isoleucine atposition 102 and an alanine at position
 106. 6. The DNA molecule ofclaim 2, wherein said DNA molecule is derived from Zea mays and ismodified to encode for said glyphosate resistant class I EPSPS proteincomprising an leucine at position 102 and an alanine at position
 106. 7.The DNA molecule of claim 2, wherein said DNA molecule is selected fromthe group consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39 and SEQ ID NO:44.
 8. The DNA molecule of claim 1, wherein said DNAmolecule is derived from a bacteria genome.
 9. A DNA constructcomprising a promoter that functions in plant cells operably linked to aDNA molecule that encodes a glyphosate resistant class I EPSPS protein,wherein said glyphosate resistant class I EPSPS protein comprises apolypeptide sequence GX₄X₁X₂RX₃, where X₁ and X₂ are any amino acid, andX₄ is isoleucine or leucine and X₃ is selected from the group consistingof threonine, glycine, cysteine, alanine, and isoleucine.
 10. The DNAconstruct of claim 9, comprising a DNA molecule selected from the groupconsisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 andSEQ ID NO:44.
 11. A method of preparing a glyphosate tolerant plantcomprising the steps of 1) contacting a recipient plant cell with theDNA construct of claim 9, wherein said DNA construct is incorporatedinto the genome of the recipient plant cell; and 2) regenerating therecipient plant cell into a plant; and 3) applying an effective dose ofglyphosate to the plant, wherein the plant displays a glyphosatetolerant phenotype.
 12. In the method of claim 11, wherein said DNAconstruct comprising a DNA molecule selected from the group consistingof SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ IDNO:44.
 13. A glyphosate tolerant plant and progeny thereof comprisingthe DNA construct of claim
 9. 14. A glyphosate tolerant plant of claim13, wherein the DNA construct comprises an EPSPS coding sequenceselected from the group consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39 and SEQ ID NO:44.
 15. A method of controlling weedsin a field of glyphosate tolerant crop plants comprising applying tosaid field of glyphosate tolerant crop plant an effective dose of aglyphosate containing herbicide, wherein said glyphosate tolerant cropplant contains a DNA construct comprising a promoter that functions inplant cells operably linked to a DNA molecule that encodes a chloroplasttransit peptide linked to a glyphosate resistant class I EPSPS protein,wherein said glyphosate resistant class I EPSPS protein comprises apolypeptide sequence GIX₁X₂RX₃, where X₁ and X₂ are any amino acid, andX₃ is selected from the group consisting of threonine, glycine,cysteine, alanine, and isoleucine.
 16. In the method of claim 15,wherein said DNA construct comprises a DNA molecule selected from thegroup consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39 and SEQ ID NO:44.
 17. A purified EPSPS enzyme comprising apolypeptide sequence GX₄X_(I)X₂RX₃, where X₁ and X₂ are any amino acid,X₄ is isoleucine or leucine, and X₃ is selected from the groupconsisting of threonine, glycine, cysteine, alanine, and isoleucine.